Eastern equine encephalitis virus (EEEV) is a mosquito-borne RNA virus endemic to the eastern United States. Due to its extreme virulence (up to 70% mortality in humans) and potential as a bioweapon, EEEV is characterized as a NIH Category B Priority Pathogen and a Select Agent. However, there are no currently licensed vaccines or antiviral therapeutics. There is very little knowledge on the role of the adaptive immune response during EEEV infection. An initial high white blood count in the cerebrospinal fluid and a shorter prodrome (e.g. fever and malaise) are indicative of a poor outcome in humans. Our lab has identified two virulence factors that contribute to the virulence of EEEV in mice and likely in humans: 1) a myeloid cell-specific microRNA, miR-142-3p, binds to the EEEV genome preventing virus replication in all myeloid cells, and 2) EEEV binds to a proteoglycan, heparan sulfate (HS), limiting viral access to peripheral lymphoid tissues. Both of these virulence factors contribute to suppression of virus replication in lymphoid tissues and suppression of the innate immune response. Mutation of the miR- 142-3p binding sites and HS binding domain resulted in myeloid cell replication in lymph nodes, production of type I interferon, and attenuation in mice. Myeloid cells in peripheral lymphoid tissues are known coordinators of the adaptive immune response through antigen presentation, costimulation, and production of signal 3 cytokines, IL-12 and IFN-?. In lymph nodes, the inability of EEEV to replicate in myeloid cells prevents the production of IL-12 and IFN-? suggesting that without the proper inflammatory milieu, the adaptive immune response may not properly develop during an EEEV infection potentially contributing to the severity of EEEV disease. The experiments in this proposal will investigate the role of virus replication in myeloid cells and lymphoid tissues on the generation of an epitope- specific CD4+ and CD8+ T cell response during an EEEV infection. Also, we will also examine whether IL-12 or IFN-? is required for signal 3 cytokine stimulation and the generation of effector CD8+ T cells in EEEV-infected mice. Understanding how the immune system develops during an EEEV infection will provide valuable information to further develop potential therapeutic interventions or novel vaccines.